1. Field of the Invention
This invention relates to a transgenic rice line containing a high level of various flavonoids in kernels of rice. Further, this invention relates to a transgenic rice line transformed with maize C1 and R-S genes.
2. Description of Prior Art
Extensive studies on crop plants have significantly improved productivity through breeding for high yield, disease/pest-resistance, and semi-dwarfism. In recent years, genetic engineering tools along with conventional breeding methods are readily employed to develop new crop varieties with not only improved productivity but also fortified nutritional quality.
One of the well-known examples is the provitamin A-producing “Golden Rice,” of which endosperm is genetically engineered with three genes involved in the carotenoid biosynthetic pathway (Ye et al., 2000). Cahoon et al. (2003) reported development of a transgenic corn that produces four to six times more vitamin E in embryos by over-expressing barley homogentisic acid geranylgeranyl transferase.
Over 2,000 flavonoids produced in plants are known to be involved in pigmentation of flowers and fruits, protection against UV and pathogens, signaling to symbiotic microorganisms, and male fertility in some plant species (reviewed in Shirley, 1996; Winkel-Shirley, 2001a, 2001b and 2002). All flavonoids possess the phenylbenzopyrone flavonoid skeleton that is synthesized through sequential condensation reactions of a p-coumaroyl-CoA and three molecules of malonyl-CoA. In addition to subtle modifications of phenylbenzopyrone itself, flavonoids are diversified by addition of various moieties such as hydroxyl, methyl and sugar groups to the basic structure.
Flavonoids have been extensively studied for their biological activities including antioxidant, antiviral, anti-cancer, anti-aging, hepatoprotective effects, cancer prevention, enhancing immune system, and improving serum lipid quality that consequently lowers the risk of cardiovascular disease (Dixon and Steele, 1999; Yousef et al., 2004).
Although most health-beneficial roles are primarily associated with antioxidant property of flavonoids functioning as reducing agents, hydrogen donors, and free radical quenchers; a number of nonantioxidant activities have been also reported for inhibitory effects on carcinogenesis (Jankun et al., 1997; reviewed in Ren et al., 2003) and modulatory effects on receptors and intracellular signaling enzymes (reviewed in William et al., 2004). Pinent et al. (2004) also reported that procyanidins, oligomeric flavonoids, have antidiabetic properties, possibly through interaction with signaling components such as phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase. As a similar work, Enomoto et al. (2004) demonstrated inhibitory effect of 3-methoxy quercetin on human aldose reductase, platelet aggregation, and blood coagulation.
Among major secondary metabolic pathways in plants, flavonoid biosynthesis is the best characterized one for molecular genetics of the genes and biochemical mechanisms of the enzymes involved in the pathway (reviewed in Holton and Cornish, 1995; Winkel-Shirley, 2001a and 2001b). Members of C1 and R regulatory gene families encoding Myb-type transcription factors and basic helix-loop-helix-type transcription factors, respectively, individually or mutually activate different sets of structural genes in the flavonoid biosynthetic pathway (Quattrocchio et al., 1993; Quattrocchio et al., 1998).
Members of both gene families have been isolated from a number of monocot as well as dicot species and known to be highly conserved within the corresponding families. For this reason, some of the C1 and R family members are functionally interchangeable among different plant species (reviewed in Schijlen et al., 2004). For example, ectopic expression of both maize C1 and R was sufficient to produce anthocyanins in root, petal, and stamen tissues that normally lack anthocyanins in Arabidopsis and tobacco (Lloyd et al., 1992). Similarly, Bovy et al. (2002) reported that tomato plants transformed with both maize C1 and LC (a member of R gene family) produced anthocyanins and additional flavonoids in the leaves and in the fruit flesh, respectively. In addition to regulatory genes, enhanced production of specific flavonoids in the specific tissues of tomato fruits was demonstrated by elevated expression of biosynthetic genes for chalcone synthase, chalcone isomerase, and flavonol synthase (Verhoeyen et al., 2002).